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Title: Assessment of synthetic chalcones as cancer chemopreventive agents
Author: Jasim, H.
ISNI:       0000 0004 8507 0365
Awarding Body: Liverpool John Moores University
Current Institution: Liverpool John Moores University
Date of Award: 2019
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Cigarette smoking acts as a key source of lung cancer, where the carcinogen malondialdehyde (MDA) produced from cigarette increases the DNA damage. To reduce the carcinogenic effect of MDA researchers are using natural or synthetic compounds as chemopreventive agents to minimise or prevent cancer. In the current research a library of synthetic chalcones were investigated, which were synthesised and kindly gifted by Prof Arroo from De Montfort University (DMU). These chalcones are categorised based on their chemical structures such as alkoxyl group (17 chalcones), methylene-dioxy group (13 chalcones), and methylene-dioxin group (1 chalcone). Nuclear factor erythroid 2-related factor 2 (Nrf2)- antioxidant response element (ARE) pathway play an important role in chemoprevention due to induction of antioxidant defence gene like NQO1 by detoxifying and reducing oxidative stress. To investigate whether the chalcones can switch on NRF2-ARE pathway, 31 synthetic chalcones were evaluated for (i) their toxicity using thiazolyl blue tetrazolium bromide (MTT) assay, and (ii) chemopreventive activity by luciferase fold induction using luciferase assay through the AREc32 reporter cell line, which includes a luciferase gene linked with ARE drive gene. The results showed that twelve chalcones have induced luciferase around 2-16 fold induction as a consequence of the switch on the Nrf2-ARE pathway. Thereafter, the selected chalcones were investigated for their ability as lung chemopreventive agents against MDA through lung normal cell line MRC-5 and cancer cell line MRC-5 SV2. Firstly, the non-toxic concentrations of chalcones in MRC-5 and MRC-5 SV2 have determined by MTT assay, then the non-toxic concentration of chalcones was examined for their ability to induce NQO1 protein, as a prototype of antioxidant enzymes, in both lung cell lines using Western blot assay. The results showed that chalcones DMU-2210, 1122, 1103 and 2265 were consequently found to be capable of increasing NQO1 protein level at 24 h in both the cell lines. Also, these twelve chalcones were then examined for their ability to prevent the toxicity of MDA. The results revealed that DMU-1113, 2265, 2210, 2267, 1103, 1122 and 1119 could reduce the toxicity of MDA in MRC-5 cells. However, the DMU-1113, 1122, 2207, 1103, 1119 and 2265 were lead to inhibit the growth of MRC-5 SV2 cells in present of MDA. The chalcones capable of NQO1 induction and reduction of MDA toxicity were then chosen to continue with the project in cytogenetic and molecular biology. Specifically, chalcones DMU-2210, 1122 and 1103, were chosen as a representative selection of the different activities noted in the library as a whole. They were examined for their ability to reduce the generation of reactive oxygen species (ROS) using intracellular ROS assay and protect DNA against damage by MDA using the comet assay. The results indicated that the three chalcones could reduce ROS levels and also DNA damage by MDA. In addition, the three chalcones were examined for their ability to induce NRF2 gene expression and consequently NQO1 gene expression using qPCR protocol, with DMU-1122 and 1103 being capable of this. Finally, to verify the hypothesis that chalcones can prevent MDA induced toxicity due to the switching on of the Nrf2 pathway, NRF2 gene was knocked down. This was confirmed by qPCR and Western blot protocols through pre-treated MRC-5 cell line with chalcones then MDA, the results showed the NRF2 gene was successfully knocked down. Simultaneously, MTT assay was used to confirm the chemopreventive activity of chalcones against MDA in MRC-5 knocked down NFR2 gene. The MTT results showed that DMU-1122 and 2210 could retain the capacity to prevent MDA induced cell toxicity in the absence of NRF2. Consequently, it could be inferred that chalcones are capable of inducing other antioxidant defence pathways crucial in the prevention of MDA toxicity.
Supervisor: Ritchie, K. ; Satyajit, S. ; Moore, S. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: RC0254 Neoplasms. Tumors. Oncology (including Cancer) ; RM Therapeutics. Pharmacology